DESCRIPTION: (Adapted from Applicant?s Description) Revolutionary advancements in genome research and the ability to create genetically specific strains of mice are resulting in an exponential increase in the number of strains available for biomedical research. Freezing and storing sperm could most efficiently preserve the majority of these newly created strains, but existing methods do not work sufficiently well for all strains of mice. The long-term objective of this project is to improve methods of sperm cryopreservation for reliable recovery of pathogen free and reproductively normal offspring. The specific aims of this project are to: (1) Establish efficient freezing protocols for improved cryopreservation of sperm from different inbred strains. Various additions and substitutions of components in an existing cryoprotectant formulation will be made within a narrow range of osmolality and tested for their effect on the efficiency of successfully freezing inbred and mutant strain sperm. (2) Enhance in vitro fertilization (IVF) for gametes from different inbred strains. Various modifications of IVF such as addition of free radical scavengers to the culture medium and removal of the zona pellucida from oocytes will be tested for ability to enhance fertilization of inbred strain gametes. (3) Determine the risk of pathogen transmission through cryopreserved sperm upon recovery of offspring through IVF or intracytoplasmic sperm injection (ICSI) using sperm of infected males sent to The Jackson Laboratory for re-derivation and importation of a strain that will be diagnosed for presence of pathogens. (4) Provide quantitative assessment of reproductive function in the animals produced from frozen sperm. Females and males produced from cryopreserved sperm will be mated to ICR mice to evaluate their fertility and will be tested for reproductive behaviors, measured for plasma levels and in vitro release of reproduction-related hormones, and assessed for spermatogenesis. Neuroendocrine mechanisms that may underlie any reproductive deficits found will be identified.